LBP Mouse

What is the biological function of LBP in mouse models?

LBP is a 58-62 kDa single-chain glycoprotein member of the BPI/LBP family that plays a crucial role in the acute-phase immunologic response to gram-negative bacterial infections . As part of a family of structurally and functionally related proteins including BPI, plasma cholesteryl ester transfer protein (CETP), and phospholipid transfer protein (PLTP), mouse LBP functions by binding to lipopolysaccharide (LPS) on the outer cell wall of gram-negative bacteria . It then transfers LPS to CD14 receptors present on the surface of cells from the myeloid lineage, markedly enhancing cellular responses to LPS .

This protein transfer mechanism transforms LPS from aggregates into monomers, catalyzing the movement of LPS to cellular receptors where it can trigger immune responses including cytokine synthesis . Through this process, LBP significantly potentiates the host's ability to detect and respond to bacterial infections at very low concentrations of LPS.

How do researchers quantify LBP in mouse biological samples?

Quantifying LBP in mouse samples typically employs enzyme-linked immunosorbent assay (ELISA) techniques specifically designed for mouse LBP detection . The Mouse LBP solid-phase sandwich ELISA uses a target-specific antibody pre-coated in microplate wells to which samples are added . This antibody captures LBP from the sample, after which a second (detector) antibody binds to LBP at a different epitope, forming a sandwich complex .

The methodological approach involves:

• Addition of samples to pre-coated wells

• Binding of detector antibody to captured LBP

• Addition of enzyme-conjugated antibody that binds to the complex

• Incubation with substrate solution to produce measurable signal

• Quantification of signal intensity, which directly corresponds to LBP concentration

This technique allows precise measurement of LBP in serum, plasma, and other biological fluids, with validation criteria including sensitivity, specificity, precision, and lot-to-lot consistency .

What are the key characteristics of recombinant mouse LBP?

Recombinant mouse LBP proteins used in research typically cover amino acids Gly25-Val481 (with variations Ser102Arg, Tyr284His) and often include a C-terminal 6-His tag for purification purposes . These products demonstrate biological activity with an ED50 (effective dose for 50% response) of 0.5-3 ng/mL .

Commercial preparations are available in two primary formulations:

The carrier protein (typically BSA) enhances protein stability, increases shelf-life, and allows storage at more dilute concentrations . The carrier-free version is recommended for applications where BSA might interfere with experimental results.

How do monoclonal antibodies to mouse LBP elucidate its function in endotoxemia?

Research employing monoclonal antibodies against mouse LBP has significantly advanced our understanding of LBP's role in endotoxemia . Scientists have developed three distinct classes of rat monoclonal antibodies to murine LBP, each affecting different aspects of LBP function:

These antibodies have demonstrated that neutralization of LBP—whether by blocking LPS binding to LBP or preventing LPS/LBP complexes from binding to CD14—protects mice from LPS-induced toxicity . This research confirms LBP's critical role in innate immunity and provides valuable tools for studying the contribution of LBP in experimental endotoxemia models.

What insights have LBP knockout mice provided about innate immunity?

LBP knockout mice have yielded sometimes contradictory but illuminating results about the role of LBP in immune responses . These mice are typically generated by targeted deletion of the LBP gene and backcrossed into C57BL/6 background multiple times to ensure genetic consistency .

Studies with these knockout models have revealed:

• In vitro, plasma from LBP-deficient mice shows significantly reduced capacity to enable cellular responses to LPS, which can be restored by adding exogenous recombinant murine LBP .

• Some in vivo studies demonstrated that LBP knockout mice were resistant to endotoxemia, suggesting a protective effect of LBP deletion .

• Contradictory findings showed no significant differences in TNF-α levels in plasma from wild-type and LBP-deficient mice injected with LPS, suggesting the existence of LBP-independent mechanisms for responding to LPS .

These disparate results highlight the complexity of in vivo immune responses and suggest compensatory mechanisms may exist when LBP is absent . LBP knockout mice continue to serve as an important tool for discovering alternative mechanisms of LPS recognition and response.

What role does LBP play in adipose tissue metabolism in mice?

Recent research has uncovered an unexpected role for LBP in adipose tissue metabolism, particularly in the browning process of adipose tissue . Studies indicate that LBP negatively mediates the browning process of both mouse and human adipose tissue .

Methodological approaches to studying this relationship include:

• Using mouse embryonic fibroblasts (MEFs) as a model system, as they represent an important source of adipocytes with multi-directional differentiation potential .

• Treating MEFs with differentiation induction reagents while manipulating LBP expression through techniques such as short hairpin RNA targeting LBP (shLBP) .

• Assessing outcomes through multiple techniques including:

  • Western blot and qRT-PCR for expression of LBP, inflammatory markers, brown fat markers, and autophagy markers

  • Oil red O staining to examine lipid formation

  • Seahorse XF96 analyzer to measure oxygen consumption

  • Glucose uptake assessments

This research direction suggests that LBP may have broader physiological roles beyond immune function, potentially impacting metabolic processes relevant to obesity management .

How do researchers effectively compare LPS transfer mechanisms of LBP versus other transfer proteins?

Comparing the LPS transfer capabilities of LBP with other transfer proteins requires sophisticated methodological approaches . Researchers typically isolate bacterial membrane blebs containing radiolabeled LPS and then assess the ability of different proteins to extract and transfer this LPS.

In experimental settings:

• When bacterial membrane blebs are incubated with LBP (1 μg/ml) and soluble CD14 (sCD14, 1 μg/ml), LBP releases approximately 9% of the LPS .

• Increasing sCD14 concentration in the presence of LBP increases LPS release from the blebs, while minimal amounts of other proteins are released .

• In contrast, when blebs are incubated with phospholipid transfer protein (PLTP, 10 μg/ml) and sCD14, no significant release of LPS occurs .

These comparative studies utilize techniques including protein isolation, radiolabeling, density gradient centrifugation, and immunological depletion methods. Such approaches help distinguish the specific mechanisms and relative efficiencies of different transfer proteins in mediating immune responses to bacterial components.

What experimental considerations are important when depleting LBP from serum for control experiments?

LBP depletion from serum requires careful methodological considerations to ensure effective removal while maintaining the integrity of other serum components . The process typically involves:

• Covalent binding of anti-LBP monoclonal antibodies (such as MAb 18G4) to solid support matrices, such as hydrazide gel beads .

• Incubating antibody-coated beads with serum samples (overnight at 4°C on a rocking platform) followed by centrifugation (850 × g, 2 min, 4°C) .

• Confirmation of depletion efficiency using ELISA to measure residual LBP concentrations .

Important considerations include:

How should researchers interpret contradictory findings from different LBP mouse models?

The interpretation of contradictory findings from different LBP mouse models requires careful consideration of several methodological factors :

• Dosage effects: The dose of LPS used in experiments can significantly impact results—LBP may play different roles at low versus high LPS concentrations .

• Genetic background: Even with extensive backcrossing (e.g., 11 generations into C57BL/6 background), genetic modifiers may influence phenotypes .

• Compensatory mechanisms: The absence of LBP from development may trigger upregulation of alternative LPS recognition pathways that obscure the true role of LBP in normal physiology .

• Experimental timing: The kinetics of immune responses may differ between wild-type and LBP-deficient mice, necessitating time-course studies rather than single-timepoint measurements.

• Measurement parameters: While some studies focus on TNF-α as a readout, comprehensive assessment of multiple cytokines and cellular responses may reveal more nuanced differences.

These considerations highlight the importance of detailed experimental design, multiple complementary approaches (gene knockout, antibody neutralization, protein supplementation), and recognition that apparent contradictions often reveal biological complexity rather than experimental error.

What are emerging applications of LBP mouse models beyond endotoxemia research?

While LBP has been primarily studied in the context of endotoxemia and innate immunity, emerging research suggests broader applications for LBP mouse models :

• Metabolic research: The discovery of LBP's role in adipose tissue browning opens possibilities for studying its contribution to obesity, diabetes, and related metabolic disorders .

• Alternative LPS recognition pathways: LBP knockout mice serve as valuable tools for identifying and characterizing LBP-independent mechanisms of LPS recognition, potentially revealing new therapeutic targets .

• Microbiome interactions: Given LBP's role in recognizing bacterial components, these models may help elucidate host-microbiome interactions in contexts such as inflammatory bowel disease, liver disease, and neuroinflammation.

• Inflammatory disease models: Beyond acute endotoxemia, LBP mouse models may inform understanding of chronic inflammatory conditions and the interplay between infection and inflammation.

• Drug development: The protective effects demonstrated by anti-LBP antibodies in endotoxemia models suggest potential therapeutic applications that could be further explored using these mouse models .

As analytical techniques continue to advance, these research directions may benefit from comprehensive multi-omics approaches that can reveal the full spectrum of LBP's biological activities and interactions.